Poor Prognosis In Pancreatic Cancer Research Articles

High Mobility Group Box 1 (HMGB1) is Associated with Progression and Poor Prognosis in Pancreatic Cancer

Aim: Increased expression of High Mobility Group Protein Box 1(HMGB1) has been reported with several different tumor types, but the role of HMGB1 in pancreatic cancer is not fully understood. The aim of this study is to detect the expression of HMGB1 and evaluate the role of HMGB1 in pancreatic cancer. Methods: The expression level of HMGB1 in pancreatic cancer cell line PANC-1 was detected by western blot analysis and reverse transcription PCR. Furthermore, primary tumors tissues of 35 patients with resected pancreatic cancer(T2-4 N0-2 M0-1) were stained immune histochemically using the polyclonal anti-HMGB1 antibody. The prognostic relevance of HMGB1 expression was evaluated by univariate Kaplan-Meier among 35 patients. Results: HMGB1 is over expressed in pancreatic cancer.HMGB1 overexpression was significantly associated with clinical stage (p<0.05). Univariate Cox regression showed that the survival time was significantly different between groups with high and low expression of HMGB1, indicating that high level of HMGB1correlated with a shorter survival time (P<0.05; HR, 2.53; 95%CI, 1.14-5.64). Conclusions: HMGB1 might promote pancreatic cancer progression and HMGB1 expression was an independent prognostic indicator for patients’ survival

Suppressed expression of NDRG2 correlates with poor prognosis in pancreatic cancer

Pancreatic cancer is a highly lethal disease with a poor prognosis; the molecular mechanisms of the development of this disease have not yet been fully elucidated. N-myc downstream regulated gene 2 (NDRG2), one of the candidate tumor suppressor genes, is frequently downregulated in pancreatic cancer, but there has been little information regarding its expression in surgically resected pancreatic cancer specimens. We investigated an association between NDRG2 expression and prognosis in 69 primary resected pancreatic cancer specimens by immunohistochemistry and observed a significant association between poor prognosis and NDRG2-negative staining (P=0.038). Treatment with trichostatin A, a histone deacetylase inhibitor, predominantly up-regulated NDRG2 expression in the NDRG2 low-expressing cell lines (PANC-1, PCI-35, PK-45P, and AsPC-1). In contrast, no increased NDRG2 expression was observed after treatment with 5-aza-2′ deoxycytidine, a DNA demethylating agent, and no hypermethylation was detected in either pancreatic cancer cell lines or surgically resected specimens by methylation specific PCR. Our present results suggest that (1) NDRG2 is functioning as one of the candidate tumor-suppressor genes in pancreatic carcinogenesis, (2) epigenetic mechanisms such as histone modifications play an essential role in NDRG2 silencing, and (3) the expression of NDRG2 is an independent prognostic factor in pancreatic cancer.

Nuclear Expression of Glioma-Associated Oncogene Homolog 1 and Nuclear Factor-κB Is Associated with a Poor Prognosis of Pancreatic Cancer

Objective: We investigated the association of the hedgehog pathway with nuclear factor (NF)-κB and clinical outcomes in pancreatic cancer patients. Methods: We analyzed tissue samples for the expression of NF-κB (RelA/p65), sonic hedgehog (Shh) and glioma-associated oncogene homolog 1 (Gli1) by immunohistochemistry and investigated their expression in association with clinical outcomes. Results: Eighty-one patients with pancreatic cancer were investigated. Expression of Shh and nuclear expression of Gli1 and NF-κB were found in 63 of 66 (96%), 28 of 68 (41%) and 22 of 68 cases (32%), respectively. Nuclear Gli1 expression was closely associated with nuclear expression of NF-κB (p < 0.001). Patients with nuclear Gli1 had significantly worse prognoses than those without (median survival 7.9 vs. 13.9 months; p = 0.009). Similarly, patients with nuclear expression of NF-κB had shorter overall survival than those with negative or cytoplasmic expression of NF-κB (median survival 5.5 vs. 13.9 months; p < 0.001). Shh expression had no prognostic significance. In the multivariate analysis, NF-κB nuclear expression was closely associated with unfavorable overall survival (p = 0.02). Conclusion: Our results indicate that nuclear expression of Gli1 or NF-κB is a strong predictor of poor prognosis in pancreatic cancer. Additional investigation of the biologic significance of this association is warranted.

CEACAM6 induces epithelial-mesenchymal transition and mediates invasion and metastasis in pancreatic cancer

Pancreatic cancer is a disease with an extremely poor prognosis. The acquisition of invasion properties in pancreatic cancer is accompanied by the process of epithelial-mesenchymal transition (EMT). Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is emerging as an important determinant of the malignant phenotype in a range of cancers, including pancreatic cancer. Therefore, the aim of this study was to evaluate the potential involvement of CEACAM6 in the invasion and metastasis of pancreatic cancer cells via EMT regulation. The results of our study showed a positive association between CEACAM6 expression and poor prognosis of pancreatic cancer, differentiation and lymph node metastasis. Elevated levels of CEACAM6 in pancreatic cancer cells promoted EMT, migration and invasion in vitro and metastasis in animal models, whereas shRNA-mediated CEACAM6 knockdown had the opposite effect. Furthermore, we demonstrated that miR-29a/b/c specific for CEACAM6 could regulate its expression at the post-transcriptional level. Collectively, our findings identified CEACAM6, which is regulated by miR-29a/b/c, as an important positive regulator of EMT in pancreatic cancer offering an explanation for how elevated levels of CEACAM6 are likely to contribute to the highly metastatic phenotype of pancreatic cancer.

Targeted therapy of spontaneous murine pancreatic tumors by polymeric micelles prolongs survival and prevents peritoneal metastasis

Nanoscaled drug-loaded carriers are of particular interest for efficient tumor therapy as numerous studies have shown improved targeting and efficacy. Nevertheless, most of these studies have been performed against allograft and xenograft tumor models, which have altered microenvironment features affecting the accumulation and penetration of nanocarriers. Conversely, the evaluation of nanocarriers on genetically engineered mice, which can gradually develop clinically relevant tumors, permits the validation of their design under normal processes of immunity, angiogenesis, and inflammation. Therefore, considering the poor prognosis of pancreatic cancer, we used the elastase 1-promoted luciferase and Simian virus 40 T and t antigens transgenic mice, which develop spontaneous bioluminescent pancreatic carcinoma, and showed that long circulating micellar nanocarriers, incorporating the parent complex of oxaliplatin, inhibited the tumor growth as a result of their efficient accumulation and penetration in the tumors. The reduction of the photon flux from the endogenous tumor by the micelles correlated with the decrease of serum carbohydrate-associated antigen 19-9 marker. Micelles also reduced the incidence of metastasis and ascites, extending the survival of the transgenic mice.

CEACAM6 induces epithelial-mesenchymal transition and mediates invasion and metastasis in pancreatic cancer

Abstract Pancreatic cancer is a disease with an extremely poor prognosis. The acquisition of invasion properties in pancreatic cancer is accompanied by the process of epithelial-mesenchymal transition (EMT). Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is emerging as an important determinant of the malignant phenotype in a range of cancers, including pancreatic cancer. Therefore, the aim of this study was to evaluate the potential involvement of CEACAM6 in the invasion and metastasis of pancreatic cancer cells via EMT regulation. The results of our study showed a positive association between CEACAM6 expression and poor prognosis of pancreatic cancer, differentiation and lymph node metastasis. Elevated levels of CEACAM6 in pancreatic cancer cells promoted EMT, migration and invasion in vitro and metastasis in animal models, whereas shRNA-mediated CEACAM6 knockdown had the opposite effect. Furthermore, we demonstrated that miR-29a/b/c specific for CEACAM6 could regulate its expression at the post-transcriptional level. Collectively, our findings identified CEACAM6, which is regulated by miR-29a/b/c, as an important positive regulator of EMT in pancreatic cancer offering an explanation for how elevated levels of CEACAM6 are likely to contribute to the highly metastatic phenotype of pancreatic cancer.

Silencing of MBD1 reverses pancreatic cancer therapy resistance through inhibition of DNA damage repair

High resistance to traditional chemo- and radiotherapies contributes to the poor prognosis of pancreatic cancer (PC). Methyl-CpG binding domain protein 1 (MBD1), which plays an important role in disease progression, contributes to the drug resistance of PC cells; however, the mechanism underlying the drug resistance endowed by MBD1 remains unknown. In this study, we found that MBD1 was recruited to DNA damage sites under DNA damage conditions. Silencing of MBD1 significantly impaired activation of the DNA damage checkpoint response and inhibited DNA repair capacity. MBD1 binds mediator of DNA damage checkpoint protein 1 (MDC1), which is induced by radiation and regulates NBS1 activation in the presence of DNA damage repair. Knockdown of MBD1 significantly increased the sensitivity of cells to radiation and cisplatin (diamindichloridoplatin, DDP) in vitro. Importantly, the function of MBD1 in regulating chemoradioresistance is also partially dependent on DNA damage repair. Thus, we hypothesize that MBD1 may promote PC chemoradioresistance by regulating PC cell fate in the presence of DNA damage. Collectively, these findings reveal an important function of MBD1 in DNA repair and mediation of chemoradioresistance of cancer cells. Moreover, this study suggests that MBD1 is a promising molecular target for sensitizing resistant PC tumor cells to chemoradiotherapy.

Abstract 417: SMAD3 upregulation is a novel biomarker of poor prognosis and epithelial-mesenchymal transition in pancreatic cancer.

Abstract Pancreatic cancer has one of the worst survival rates among all cancers. Features like epithelial-mesenchymal transition (EMT) are often seen in pancreatic tumors and correlate with poor prognosis. The purpose of this study was to identify factors involved in EMT in pancreatic cancer that may serve as prognostic indicators. Gene-expression profiling identified overexpression of SMAD3 in pancreatic cancer with high-grade solitary cell infiltration, one of the EMT-like features. SMAD3 expression was then evaluated through immunohistochemical analysis using clinical specimens. SMAD3 was accumulated in the nuclei of tumor cells (positive rate ranging from 0% to 75%), but was not detected in most of the epithelial cells in the pancreatic duct. Upregulated SMAD3 (≥15% positive in nuclei of tumor cells) correlated with malignant characteristics, such as higher tumor grade (P = 0.001) and lymph node metastasis (P &amp;lt;0.001), as well as with EMT-like features, such as reduced E-cadherin (P = 0.013) and elevated vimentin (P = 0.032). SMAD3 knockdown in pancreatic cancer cells resulted in increased E-cadherin expression, decreased vimentin expression and decreased cell motility. The suppressed EMT-like features by SMAD3 knockdown were observed even in SMAD4-inactivated pancreatic cancer cells, suggesting that SMAD3 is involved in EMT induction, regardless of SMAD4 status. In clinical cases, SMAD3 upregulation and EMT-like features correlated with shorter survival time. Moreover, SMAD3 and E-cadherin were identified as independent prognostic factors by multivariate analysis, and the group of patients with both upregulated SMAD3 and reduced E-cadherin had the least favorable prognosis compared with other groups. Taken together, SMAD3 seems to play an important role in malignant progression of pancreatic cancer, and may provide a potential biomarker for poor prognosis of pancreatic cancer. Citation Format: Ken Yamazaki, Yohei Masugi, Kathryn Effendi, Michiie Sakamoto. SMAD3 upregulation is a novel biomarker of poor prognosis and epithelial-mesenchymal transition in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 417. doi:10.1158/1538-7445.AM2013-417

Abstract 3439: Suppression of pancreatic cancer cell growth by NISC-6 through activation of Par-4 and death receptor 5.

Abstract Pancreatic cancer is one of the deadliest cancers, with a median survival of 6 months and a 5 year survival rate of 3-5%. The poor prognosis of pancreatic cancer is attributable to its tendency for late presentation, aggressive local invasion, early metastases, and poor response to chemotherapy. Currently used drugs result in marginal survival advantage and are associated with multiple adverse events and drug resistance. Thus, there is a need for novel therapeutic agents and strategies involving less toxic agents that can sensitize pancreatic cancer cells to chemotherapy. Through extensive structure-activity relationship studies, we have recently developed an Akt pathway inhibitor, NISC-6, designed by incorporating an isoselenocyanate (-N=C=Se) functionality into naphthalimide structure, rendering it lethal to cancer cells at a dose that is non-toxic to normal cells. Our hypothesis is that the Akt inhibitor, NISC-6, in conjunction with Prostate apoptosis response protein 4 (Par-4), will induce apoptosis in pancreatic tumor cells through the TRAIL pathway. This is based on the observation that Par-4 causes apoptosis in cancer, but not in normal, cells, often in response to an apoptotic agent and is often inactivated in cancer cells through interaction with Akt1. In pancreatic cancer, Par-4 expression is often reduced through deletion of a chromosome segment containing 12q21, the location of the PAR-4 gene. Activation of Par-4 through inhibition of Akt by NISC-6 thus holds promise as a possible strategy for pancreatic cancer treatment. Our studies have shown NISC-6 to be effective in reducing cell proliferation in three different pancreatic cancer cell lines e.g. MiaPaCa-2, Panc-1, and BxPC-3. The treatments of NISC-6 for 48 h reduced the cell viabilities of pancreatic cancer cells with an IC50 of between 1-2.5 μM. In addition, treatment of MiaPaCa-2 cells with NISC-6 for 24 h showed significant activation of Par-4 and death receptor 5 (DR5). This activation was associated with the down-regulation of anti-apoptotic protein Bcl-xL and Survivin. Furthermore, NISC-6 potentiated the apoptotic effects of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO-2L). Taken together, these results suggest that NISC-6 may represent a novel therapeutic agent to sensitize pancreatic cancer cells to TRAIL and chemotherapeutic drugs. Detailed results of these investigations will be presented. Citation Format: Deepkamal Karelia, Manoj K. Pandey, Rosalyn Irby, Shantu Amin, Arun K. Sharma. Suppression of pancreatic cancer cell growth by NISC-6 through activation of Par-4 and death receptor 5. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3439. doi:10.1158/1538-7445.AM2013-3439

Abstract 5207: Targeting prion protein as a potential oncogene in pancreatic cancer.

Abstract Background: Human prion protein (PrP) has been found to be up-regulated in gastric, breast, pancreatic cancers and its expression in cancer cells contributes to cancer progression and resistance to various cancer therapies. Current literature suggests that overexpression of PrP induces inherent resistance to cancer therapeutics, while repression of PrP induces sensitization to cancer therapeutics. Our previous studies using expression microarray have revealed that PrP is not detected in normal human pancreatic ductal cells but is expressed in human pancreatic ductal adenocarcinoma (PDAC) cell lines and 40% of pancreatic cancer specimens. Further, expression of PrP confers a worse prognosis in PDAC patients. The poor prognosis of pancreatic cancer demonstrates needs for better target and drug development. Our recent studies also found that PrP is universally expressed in fetal pancreatic precursor ductal cells, and co-expresses with pancreatic stem cell markers, such as Pdx1 and Notch1, suggesting that PrP is a marker of pancreatic stem cells and may cross-talk with Notch1 during development and pancreatic tumorigenesis. However, the role for PrP in the establishment or progression of cancer is unknown, and whether PrP cross-talks with oncogenes such as Notch in these processes need to be elucidated. Methods: We applied immunoprecipitation (Abcam, Anti-Prion protein antibody 8H4) and western blotting to investigate the binding partners for PrP. Knockdown and over-expression of PRNP gene were carried out on various pancreatic cell lines, followed by in vitro proliferation assay and apoptotic activity analysis of these cells. Results: Pancreatic cancer cell lines express different levels of PrP, e.g. BxPC3 and Pan02.03 have higher PrP expression when compared to Capan-1. Western blot analysis revealed that PrP can bind with Notch1 protein in both BxPC-3 and CAPAN-1 cell lines. Moreover, down-regulation of PrP led to decreased cell proliferation and accelerated apoptosis, accompanied by down-regulation of cleaved/activated Notch1 (ICN1) and Notch2 (ICN2) expression in BxPC3 cells, but had lesser effect on CAPAN-1 cells. In comparison, over-expression of PrP significantly increased the proliferation rate of CAPAN-1 cells and dramatically upregulated the expression of ICN1. In conjunction with our previous findings that PrP exists as a pro-prion that binds filamin A in pancreatic cancer cells, these findings suggest that PrP may mediate pancreatic cancer cell progression through regulation of Notch activity and other cell mechanic and signaling integrators. This hypothesis is being further tested by xenograft analysis. Elucidating the mechanisms for PrP mediated Notch activation in PDAC will further our long-term goal of understanding the role of oncogenic PrP in conferring pancreatic cancer aggressiveness, and potentially will lead to novel therapeutic options for treatment of pancreatic cancer. Citation Format: Yiwei Wang, Lan Zhou, Wei Xin. Targeting prion protein as a potential oncogene in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5207. doi:10.1158/1538-7445.AM2013-5207

α-Mangostin suppresses lipopolysaccharide-induced invasion by inhibiting matrix metalloproteinase-2/9 and increasing E-cadherin expression through extracellular signal-regulated kinase signaling in pancreatic cancer cells

Invasion and metastasis are major factors in the poor prognosis of pancreatic cancer, which remains one of the most aggressive and lethal diseases worldwide. α-mangostin, a major xanthone compound identified in the pericarp of mangosteen (Garcinia mangostana, Linn; GML), possesses unique biological activities, including antioxidant, antitumor and anti-inflammatory effects. Whether α-mangostin is able to inhibit the invasive ability of pancreatic cancer cells has not been elucidated. In the present study, α-mangostin was shown to inhibit the invasive ability of the pancreatic cancer cell lines MIAPaCa-2 and BxPC-3. The results showed that α-mangostin inhibited the growth of the pancreatic cancer cells in a dose- and time-dependent manner. At concentrations of <5 μM, α-mangostin had no significant effects on cytotoxicity, but significantly inhibited the invasion and migration of pancreatic cancer cells and the expression of matrix metalloproteinase (MMP)-2 and MMP-9, while increasing the expression of E-cadherin. The present data also showed that α-mangostin exerted an inhibitory effect on the phosphorylation of extracellular-signal-regulated kinase (ERK). Furthermore, the reduction of ERK phosphorylation by small interfering RNA (siRNA) potentiated the effect of α-mangostin. Taken together, the data suggest that α-mangostin inhibited the invasion and metastasis of pancreatic cancer cells by reducing MMP-2 and MMP-9 expression, increasing E-cadherin expression and suppressing the ERK signaling pathway. The present study suggests that α-mangostin may be a promising agent against pancreatic cancer.

Anticancer effects of Garcinol in pancreatic cancer transgenic mouse model

Poor prognosis in Pancreatic cancer (PC) is attributable to weak diagnostic and treatment options. Surgical resection is possible for 20% of diagnosed cases while current chemotherapy (Gemcitabine) has a 15% response rate. Previously, we reported that Garcinol, a polyisoprenylated benzophenone, from Garcinia indica, shows a favorable dose dependant response in PC cells, alone and in combination with Gemcitabine. In this study we report the invivo effects of dietary Garcinol in transgenic pancreatic cancer mouse model (KPC).MethodsKPC (k‐ras and p53 conditional mutant) mice were divided into 4 groups: KC‐cancer control; KGr− 0.05% Garcinol diet; KGm‐Gemcitabine injected; KGG− Garcinol+Gemcitabine. Littermates without KPC mutations served as controls. PC progression was monitored by MRI and confirmed by tissues stained with Hematoxylin and Eosin. Blood smears were probed for changes in lymphocytes.ResultsThe KGr group showed improved survival rate. MRI scans showed reduction in tumor volume in KGr, KGm and KGG groups. This was supported by the reduced number of Pancreatic Intraepithelial Neoplasia 3 (PanIN 3) in KGr and KGG groups as compared to the KC group. In addition, an increase in NK (natural killer) cells was observed in KGr group.Taken together, our data indicates that dietary Garcinol decreased pancreatic cancer progression in transgenic pancreatic cancer mice.Funded by Intramural funds.Grant Funding Source: Intramural funds

Masitinib in nonresectable pancreatic cancer: Results of a phase III randomized placebo-controlled trial.

158 Background: Masitinib is a selective c-Kit inhibitor that efficiently inhibits mast cell function. Increased mast cell activity in the tumor microenvironment has been linked to poor prognosis in pancreatic cancer (PC) patients. Methods: In a randomized (1:1 ratio), double-blind, international phase III trial, chemo-naïve PC patients (n=348) received either masitinib (9 mg/kg/day) in combination with gemcitabine (1000 mg/m2/wk) (M+G) or placebo plus gemcitabine (P+G). Primary endpoint was overall survival (OS). An ancillary pharmacogenomic study, based on RNA extracted from whole blood prior to treatment, was conducted in a subset of patients. Results: In the overall population median OS was not significantly improved in the M+G arm as compared with the P+G arm (median OS: 7.7 vs. 7.0 months, respectively; p=0.74, HR=0.90 [0.71; 1.14]). However, M+G treatment led to a significant survival advantage in two subpopulations. First, in patients with ‘pain’ at baseline (defined as a VAS score &gt;20 mm on a 100 mm scale) (44% of patients with pain intensity data available) median OS was significantly increased from 5.4 months in the P+G arm to 8.1 months in the M+G arm (p=0.010; HR:0.61 [0.42; 0.88]); OS rates at 12 and 18 months were respectively, 32.2% and 18.2% in the M+G arm vs. 17.8% and 7.8% in the P+G arm. Second, in patients with a specific deleterious genomic biomarker (GBM) consisting of a limited number of genes (66% of patients with genetic data available), median OS was significantly increased in the M+G arm as compared with the P+G arm (11.0 vs. 5.0 months, respectively) (p=0.000038; HR=0.29 [0.17; 0.51]); OS rates at 12 and 18 months were respectively, 41.4% and 18.5% in the M+G arm vs. 11.1% and 4.2% in the P+G arm. In patients without pain (defined as VAS &lt;5 mm and no need for opioid analgesics), median OS was 15.4 months in the P+G arm. In patients without the deleterious GBM, median OS in the P+G arm was 14.3 months. In these two patient populations M+G treatment was contraindicated. The general safety profile of the M+G combination regimen was acceptable. Conclusions: Masitinib in combination with gemcitabine provides a significant survival advantage over gemcitabine monotherapy in PC patients with ‘pain’ and patients with a specific GBM. Clinical trial information: NCT00789633.

Preoperative chemoradiotherapy, surgery and adjuvant therapy for resectable pancreatic cancer.

In order to improve the poor prognosis of pancreatic cancer, a combination therapy consisting of preoperative chemoradiotherapy, surgery and postoperative chemotherapy may be an ideal strategy; nevertheless, the influence of preoperative therapy to postoperative therapy is not investigated. Thirty patients with resectable pancreatic ductal adenocarcinoma were enrolled. A 40Gy of radiation (2Gy/day x 20 fractions/4 weeks) was administered together with intravenous infusion of gemcitabine (800mg/m2, days 1, 8 and 15) before surgery. Surgery was performed 3-7 weeks after the final fraction of radiation, and postoperative chemotherapy consisting of 1000mg/m2 gemcitabine (days 1, 8 and 15 every 4 weeks for 6 cycles) was started within 8 weeks after surgery. All 30 patients successfully completed preoperative therapy. Re-staging after such therapy showed radiologically unresectable disease in 4 patients and 1 patient rejected surgery. Among the 25 patients who underwent laparotomy, 21 underwent curative resection. After curative resection, 4 were inadequate in performance status, thus postoperative therapy could not be started. Ten patients completed postoperative adjuvant therapy. The combination therapy for resectable pancreatic cancer seems a feasible and effective approach, though preoperative therapy may reduce the feasibility of postoperative therapy.

Novel therapeutic strategies targeting tumor-stromal interactions in pancreatic cancer

Therapy-resistance and postoperative recurrence are causes of the poor prognosis in pancreatic cancer. Conventional therapies have a limited impact on the control of pancreatic cancer, resulting in the rapid re-growth of the tumor. The indispensable role of tumor-stromal interaction, which acts as a defender of cancer cells and enhances malignant potential, is being uncovered now. For example, specific signaling pathways for desmoplasia induction have been identified, such as sonic hedgehog (Shh) or connective tissue growth factor (CTGF), whose inhibition causes desmoplasia depletion and therapeutic advantages at least in in vivo mouse models of pancreatic cancer. Revolutions in drug delivery methods have led to the establishment of novel chemotherapeutic regimens, with better patient survival. Furthermore, mechanisms of immunosuppression in the pancreatic cancer-bearing host were clarified by the identification of myeloid-derived suppressor cells (MDSCs), which also promote disease progression. Strategies to target these components of the tumor stroma revealed certain anticancer effects in vitro and in vivo, suggesting the possibility of stroma-targeting therapy. Suppression of the stromal cell function increases the sensitivity of pancreatic cancer cells to therapeutic intervention. Further study will clarify the complex nature of the tumor microenvironment, the targeting of which has the potential to improve clinical outcome.

Abstract P1-05-13: CLIC3 is associated with invasive behaviour and poorer prognosis in estrogen receptor-negative breast cancer.

Abstract Background: We previously described a Chloride Intracellular Channel-3 (CLIC3)-dependent recycling pathway which trafficked active integrins from late endosomes to the cell surface and which was required for the migratory and invasive behaviour of A2780 ovarian cancer cells in vitro. We also demonstrated that elevated expression of CLIC3 was associated with poor prognosis in pancreatic cancer. We therefore set out to investigate the role of CLIC3 in breast cancer. Materials and Methods: CLIC3 expression was assessed by immunohistochemistry and the weighted histoscore method in a tissue microarray (TMA) consisting of triplicate cores from 141 patients diagnosed with invasive estrogen receptor (ER)-negative early breast carcinoma between 1995 and 1998. Full clinicopathological and follow-up data were available. Further data were obtained from publicly available gene expression datasets (Desmedt, GSE7390) and Oncomine™. Knockdown of CLIC3 in the ER-ve MDA-MB231 breast cancer cell line was achieved by nucleofection of 2 different siRNA sequences (Dharmacon). Inverse invasion assays measured invasion into a plug of matrigel supplemented with fibronectin over 72 hours whilst organotypic invasion assays measured invasion into a fibroblast-containing collagen matrix over 6 days. Results: CLIC3 mRNA expression was significantly elevated in breast cancer in comparison to normal breast tissue in two independent data sets. High CLIC3 protein levels were associated with significantly shorter breast cancer specific survival (p = 0.026) in a TMA of 141 ER-ve patients. This finding was corroborated by the observation that high CLIC3 mRNA levels were associated with shorter overall survival in 198 patients in the Desmedt dataset (p = 0.038). Transient silencing of CLIC3 expression using two independent siRNA sequences had no effect on proliferation of MDA-MB231 cells but significantly reduced their invasiveness by 46% and 93% respectively in an inverse invasion assay and by 36% and 42% respectively in an organotypic invasion assay. CLIC3 was found to co-localise with Rab7 and LAMP1 in late endosomes/lysosomes in MDA-MB231 cells. Conclusions: Our clinical and in vitro data indicate an important role for CLIC3 in the invasive and metastatic behaviour of some breast cancers. Further work to elucidate molecular mechanisms is underway and will be presented. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-13.

The clinical significance of SWI/SNF complex in pancreatic cancer

Chromatin remodeling factors have been the subject of great interest in oncology. However, little is known about their role in pancreatic cancer. The objective of this study was to clarify the clinical significance of the SWItch/sucrose nonfermentable (SWI/SNF) complex in patients with pancreatic cancer. A total of 68 patients with pancreatic cancer who underwent R0, 1 resection were enrolled. Cancer tissues were processed to tissue microarray, then stained immunohistochemically by using antibody of SWI/SNF components; BRM, BRG1, BAF250a, BAF180 and BAF47. The correlation of expression levels and clinicopathological outcomes were analyzed, followed by the multivariate analysis of prognostic factors for overall survival. The expression levels of the SWI/SNF components were categorized as low or high according to the median value of Histoscore. Statistical analysis revealed that BRM expression was related to tumor size, T factor, M factor, lymphatic invasion and stage BRG1 expression to histology and stage BAF180 expression to tumor size and BAF47 expression to lymphatic invasion, respectively. Multivariate Cox proportional hazard analysis showed that high BRM and low BAF180 expression levels were independent predictors of worse survival in patients with pancreatic cancer. High BRM, and low BAF180 were also independent prognostic factors for poor survival in the subgroup with adjuvant gemcitabine. These results suggest that the specific cofactors of SWI/SNF chromatin remodeling complex certainly have roles in pancreatic cancer. High BRM, and low BAF180 are useful biomarkers for poor prognosis in pancreatic cancer.

Reduced expression of Raf kinase inhibitor protein correlates with poor prognosis in pancreatic cancer

Raf kinase inhibitory protein (RKIP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. But its function in pancreatic cancer was not yet clarified completely. The aim of this study was to investigate the involvement of RKIP in pancreatic cancer. RKIP expression was investigated retrospectively by immunohistochemistry in paraffin-embedded primary tumor tissue samples from a series (n = 99) of consecutive patients with pancreatic cancer. Survival was calculated using Kaplan-Meier curves. Parameters found to be of prognostic significance in univariate analysis were verified in a multivariate Cox regression model. RKIP expression was high in normal pancreatic epithelium and retained to varying degrees in pancreatic cancer tissues. However, in tumor tissues with lymph node metastasis (P = 0.008) and high UICC stage (P = 0.006), RKIP expression was highly significantly reduced or lost. Furthermore, the reduced expression of RKIP significantly correlated with both poor overall and disease-free survival (P = 0.008 and 0.01, respectively). Multivariate analyses revealed RKIP to be an independent prognosticator. These findings suggest that RKIP could be a promising marker for predicting a better prognosis in pancreatic cancer.

L-Carnitine-supplementation in advanced pancreatic cancer (CARPAN) - a randomized multicentre trial

BackgroundCachexia, a >10% loss of body-weight, is one factor determining the poor prognosis of pancreatic cancer. Deficiency of L-Carnitine has been proposed to cause cancer cachexia.FindingsWe screened 152 and enrolled 72 patients suffering from advanced pancreatic cancer in a prospective, multi-centre, placebo-controlled, randomized and double-blinded trial to receive oral L-Carnitine (4 g) or placebo for 12 weeks. At entry patients reported a mean weight loss of 12 ± 2,5 (SEM) kg. During treatment body-mass-index increased by 3,4 ± 1,4% under L-Carnitine and decreased (−1,5 ± 1,4%) in controls (p < 0,05). Moreover, nutritional status (body cell mass, body fat) and quality-of-life parameters improved under L-Carnitine. There was a trend towards an increased overall survival in the L-Carnitine group (median 519 ± 50 d versus 399 ± 43 d, not significant) and towards a reduced hospital-stay (36 ± 4d versus 41 ± 9d,n.s.).ConclusionWhile these data are preliminary and need confirmation they indicate that patients with pancreatic cancer may have a clinically relevant benefit from the inexpensive and well tolerated oral supplementation of L-Carnitine.

P-0100 H4K12 And H3K18 Acetylation Associates with poor Prognosis in Pancreatic Cancer

IntroductionEpigenetic deregulation may be involved in tumor cell biology, including cell growth, differentiation, tumor progression and cell death, and histone acetylation is a major regulatory mechanism of gene transcription. Patterns of global histone modifications have been recently suggested as outcome predictors in cancer patients, but few studies have been conducted on pancreatic ductal adenocarcinomas (PDACs). This study was designed to investigate the predictive value of histone acetylation modifications in pancreatic cancer. MethodsA retrospective clinicopathologic analysis was undertaken in all patients diagnosed with PDAC between 2005 and 2011 at our institution, and immunohistochemistry performed with polyclonal antibodies against H4K12ac, H3K9ac and H3K18ac. Semi-quantitative assessment of the percentage of tumor cells with positive nuclear staining (0% to 100%) and intensity of staining (0 to 3+) was performed independently by two pathologists who were blinded to all clinicopathologic and outcome variables. Immunohistochemical staining was also evaluated by the modified Histo-score (H-score), which involves assessment of both the intensity of staining and the percentage of positive cells. Nuclear staining for each histone was further classified into low or high-staining groups. Results were analyzed in relation to patients' clinicopathologic parameters. ResultsA total of 119 patients with pancreatic cancer were included in this study. The median age at diagnosis was 66 years (range, 36-92 years, SD±10.3) and median post-diagnosis follow-up was 14.2 months. Ninety patients (76%) died during the follow-up period. Median percentage of cells staining positive for H4K12ac, H3K9Ac and H3K18ac was 60% (±26.9), 80% (±21.2) and 65% (±24.7), respectively. There was a positive relationship between tumor differentiation and H4K12ac high scores (p<0.05) and staining with the three markers correlated positively with tumor stage (p <0.01). Univariate analysis showed worse survival in patients with high detection levels of H4K12ac (p=0.038) and H3K18Ac (p = 0.033). A backwards Cox proportional hazards model analysis revealed the independent prognostic effect of high H4K12ac and H3K18ac levels (hazard ratios of 1.6 and 1.7 respectively, p <0.05), especially for patients at early stages of disease (I and II). ConclusionHere we demonstrate that two histones modifications, H4K12 and H3K18 acetylation, correlates with worse prognosis and poor survival in pancreatic cancer, being independent prognostic factors for overall survival. The prognostic role of H3K9ac remains elusive, mandating further evaluation. Additional studies of histone modifications in pancreatic cancer can lead to a better comprehension of the epigenetic mechanisms involved in carcinogenesis and provide the information necessary to develop early diagnosis and therapeutics strategies.